This single-center randomized clinical trial was conducted in the Siping Hospital of China Medical University (Jilin, China) between January, 2011 and January, 2014. The aim of the present study was to assess the efficacy of hUC-MSCs grafting by intra-arterial infusion for the treatment of early-stage ONFH. The protocol of the present study was approved by the Institutional Review Board and the Ethics Committee of Siping Hospital of China Medical University. Written informed consent was obtained from each patient before enrollment.

Patients were evaluated clinically and radiologically using the Association Research Circulation Osseous (ARCO) classification (18) and the Harris hip score (HHS) (19) pre-operatively and at 12 and 24 months post-operation. All the patients in this study were ≥18 years of age, and were classified as ARCO stages II to IIIa. The patients had femoral head collapse of <2 mm and no evidence any damage to the acetabular cartilage. Those patients who were excluded from this study had damages caused by a traumatic event or experienced severe hemorrhage. We also excluded those who were not available for follow up because of life-threatening conditions or had missing stratification information. Those without standard care and rehabilitation were also excluded. This exclusion criterion was chosen to ensure that all the patients were treated according to the best medical practice.

We included 9 patients (9 hips) for this study. The general characteristics of the patients are shown in Table I. Blood was taken from the internal jugular veins. The outcomes were evaluated based on the HHS and the radiological results.

Six human equally sized UC were collected after informed consent was obtained from mothers in accordance with the Ethics Committee of the Institute of Siping Hospital of China Medical University. Informed consent was obtained from all the cases. Experiments and laboratory procedures were carried out in the Siping Hospital of China Medical University. From each sample, sections of 8–10 cm of the UCs were internally washed with phosphate-buffered saline (PBS) containing 300 U/ml penicillin and 0.3 mg/ml streptomycin and immediately immersed in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) supplemented with 10% AB-human serum (all from Gibco, Grand Island, NY, USA), 300 U/ml penicillin, and 0.3 mg/ml streptomycin. The samples were processed within 12–15 h after collection.

UCs were filled with 0.1% collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS and incubated at 37°C for 20 min. Each UC was washed with proliferation medium (a-MEM + 10% AB-human serum), and the detached cells were harvested after gentle massage of the UC. The cells were centrifuged at 300 × g for 10 min, resuspended in proliferation medium, and seeded in 75-cm² flasks at a density of 5×10⁷ cells/ml. After 24 h of incubation, non-adherent cells were removed, and culture medium was replaced every 3 days. Adherent cells were cultured until they reached 80–90% confluence. The cells were observed using a phase contrast microscope (Olympus, Tokyo, Japan).

To analyze the cell-surface expression of typical protein markers, adherent cells were passaged with 0.25% trypsin (Gibco). The cells were washed in PBS and fixed in a 4% paraformaldehyde solution (both from Sigma-Aldrich, Milwaukee, WI, USA) for 10 min; and incubated with the following anti-human primary antibodies: mouse monoclonal CD45-phycoerythrin (PE) (Abcam, Cambridge, MA, USA; catalog no.: ab25603; dilution: 0.2 µg/10⁶ cells), mouse monoclonal CD31-fluorescein isothiocyanate (FITC) (Abcam; catalog no.: ab33858; dilution:10 µl/10⁶ cells), mouse monoclonal CD90-FITC (Abcam; catalog no.: ab11155; dilution: 10 µl/10⁶ cells), mouse monoclonal HLA-DR-PE (Abcam; catalog no.: ab95830; dilution: 5 µl/10⁶ cells). A total of 10,000 labeled cells were analyzed using a Guava easyCyte flow cytometer running Guava Express Plus software (Guava Technologies, Inc., Chicago, IL, USA).

For osteocyte differentiation, the cells were plated at 10⁴ cells/cm² on uncoated plastic (Permanox) chamber slides (Lab-Tek; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and allowed to adhere for 24 h in tissue culture media (DMEM-LG and 10% human AB serum), after which the media were replaced with osteogenic induction media, composed of DMEM-LG supplemented with 10% human AB serum, 1% penicillin/streptomycin, 50 µg/ml L-ascorbic acid (Wako Chemicals GmbH, Neuss, Germany), 10 mM glycerol phosphate disodium salt (β-glycerophosphate), 10 nM dexamethasone, and 10 nM calcitriol (1α,25-dihydroxyvitamin D3) (Sigma, Irvine, UK).

Alizarin Red S (ARS) staining was used to examine osteogenic differentiation and mineralization. Specimens were washed with PBS, fixed with 10% formaldehyde, and stained with ARS (Millipore Corp., Billerica, MA, USA), which stained calcium minerals into a red color.

Red blood cell-related parameters were analyzed as blood cells before operation and third day after operation using an automated blood cell counter LH-750 (Beckman Coulter, Inc., Brea, CA, USA).

MRI was performed using Discovery MR750 3.0T (GE Medical Systems, Milwaukee, WI, USA) before and 12/24 months after intra-arterial infusion with coronal T1-weighted imaging, T2-weighted imaging and axial T1-weighted imaging, and T2-weighted imaging. Images (3-mm) with a 0.5-mm gap were obtained using a 256×192 matrix and four excitations. The volumetric analysis of the ONFH was assayed by Syngo via 2.0 (GE Medical Systems, Milwaukee, WI, USA).

Under general anesthesia, each patient was placed in the supine position, and the right femoral artery was punctured using the Seldinger technique with a 4.0 F Cobra catheter (Terumo, Tokyo, Japan). MSCs (10 ml) with a cell density of 5×10⁶ to 1×10⁷/ml were intra-arterially injected. Once the needle was fully withdrawn, the puncture site was wrapped with sterilized dressing. The patients remained in the supine decubitus on the operation bed for another 30 min before being returned to individual wards. Antibiotics were given to prevent infection.

The patients were instructed to be non-weight-bearing for 4 weeks and partial weight-bearing for the subsequent 6 weeks. Full weight-bearing was achieved 6 months postoperatively (20).

ODI was calculated as the ratio between the hematocrit and systolic blood viscosity (SBV) (21,22). ODI = hematocrit/SBV = 100H/1.4175 + 5.878H-12.98H² + 31.964H³, where H is the volume fraction of erythrocytes.

The following treatment protocol was used: i) anti-platelet aggregation drugs: sarpogrelate (Mitsubishi Tanabe Pharma Corp, Tianjin, China) 100 mg t.i.d. + clopidogrel hydrogen sulfate (Salubris Co., Ltd., Shenzhen, China) 50 mg/day. According to the target lesions run-off situation, dual anti-platelet drugs should continued one year post-operation at least; ii) medications for improving circulation: alprostadil injection (Tide Pharmaceutical Co., Ltd., Beijing, China); and iii) perioperative anticoagulation drugs. Low molecular weight heparin sodium injection 4000 iu q12h hypodermic injection (Clexane; Aventis Intercontinental, Antony, France) was also used.

The evaluations consisted of clinical and radiographic analysis preoperatively and at the end of follow-up based on the HHS. All the patients underwent clinical and radiographic examinations at 12 and 24 months post-operation. An excellent or good HHS was defined as ≥80 points and scores <70 were considered negative. Analyses were completed using SPSS version 22.0 software (IBM SPSS, New York, NY, USA).

The general characteristics of patients are shown in Table I. The patients included 4 males and 5 females, with an age range of 28–51 years (mean, 41.13±3.29 years). The etiology of the osteonecrosis was corticosteroid use in 6 hips, intemperance in 2 hips, and idiopathic or unknown in 1 hips; 5 hips had stage II osteonecrosis and 4 had stage IIIa osteonecrosis.

Cells derived from UC were observed 24 h after they were seeded (Fig. 1A), when part of the round mononuclear cells was adherent. Three days after inoculation, small colonies of the adherent cells with typical fibroblast-shaped morphology were obtained (Fig. 1B). These primary cells reached monolayer confluence, after planting for 5–6 days, when passaged for the first time. Fifth-passaged cells were analyzed by flow cytometry, and were strongly positive for CD31 (98.95%) and CD90 (97.77%), but negative for CD45 (0.55%) and HLA-DR (0.14%) (Fig. 1C and D).

ARS staining was used to examine osteogenic mineralization. The purpose of this method was to confirm that hUC-MSCs cultured in osteogenic media were able to produce minerals. ARS stained calcium minerals into a red color (Fig. 2), while no red staining was found in hUC-MSCs cultured in tissue culture media, which served as a control (data not shown). hUC-MSCs in osteogenic media showed significant mineral staining from the 7th day (Fig. 2). Red staining became progressively thicker and darker at 14 and 21 days.

The pre-operation red cell count, Hb and Hct levels were significantly reduced three days after the operation. The same results were observed in the mean cell volume, mean cell Hb concentration, mean cell Hb and red cell distribution width. Related parameters of platelet remained unchanged at all time points, as anti-platelet aggregation drugs were used before and after the operation (Table II).

As shown in Table III, after operation, ODI in all the patients increased by about 5% compared to pre-operation readings. This increase was especially more obvious in the ARCO stage II patients. These results consisted of Hct reduction, which suggested a more efficient oxygen transport (23).

MRI was performed to determine conditions of ONFH after 12 months. The patients showed sporadic low signal intensity on T1W, but no obvious abnormal signal intensity on T2W R, profile rules, and comparatively smooth edge (Fig. 3B). The result from the volumetric analysis showed that the necrotic volume of femoral heads was 7.16±0.73 cm³; whereas, the necrosis volume at 24 months decreased to 5.86±1.67 cm³ (p<0.05) (Fig. 3C).

The mean preoperative HSS was 39.19±5.06 points pre-operation (Table IV). HHS points increased obviously at the end of the 12 month postoperation, but these points decreased 10% by the end of the 24-month post-operation.